DNA is continuously exposed to environmental as well as endogenous factors that threaten overall genome integrity. Some of the primary contributors to DNA damage are lesions affecting one strand of the DNA, or single template lesions (STLs). These come in a variety of manifestations including alkylated bases, UV-induced dimers, single strand nicks, and secondary DNA structure (e.g. hairpins and G4 DNA). The necessity for DNA replication machinery to bypass and repair these lesions is of utmost importance, as failure to do so can result in mutagenesis and inappropriate recombination that ultimately leads to cancer or cellular senescence. To date, a number of different DNA repair pathways have been implemented in bypass and repair of STLs, including five Homologous Recombination (HR)-dependent pathways: Double Strand Break Repair (DSBR), Break Induced Replication (BIR), Fork Reversal (FR), Template Switch Recombination (TSR), and Gap Repair (GR). Each of these pathways employ specific recombination intermediates (RIs), with the former three pathways utilizing single or double Holliday Junctions (HJs or dHJs), and the latter two pathways utilizing Rec-Xs. The goal of this proposal is to use biochemical characterizations of RIs to determine which pathways respond to specific types of lesions, and which factors are responsible for the formation and resolution of RIs within these pathways. My first specific aim is to use a combination of genetic manipulation and drug treatment in yeast to determine conditions in which only one RI predominates. My second aim will utilize novel synthetic DNA substrates to identify factors responsible for the resolution of specific RIs. Together, these aims will provide a better understanding of how the many HR-pathways function together to repair specific STLs, and how these pathways can be better targeted to treat cancers.